2.1 Cancer
In the United States, cancer accounts for over 500,000 deaths annually, a toll second only to that from cardiovascular diseases. Current statistics suggest that approximately 30 percent of Americans will develop cancer within their lifetime, of whom about two-thirds will die as a result of their disease.
Cancer is not fully understood on the molecular level. It is known that exposure of a cell to a carcinogen, such as certain viruses, certain chemicals or radiation, leads to DNA alteration that inactivates a “suppressive” gene or activates an “oncogene”. Suppressive genes are growth regulatory genes which, upon mutation, can no longer control cell growth. Oncogenes are initially normal genes (called protoncogenes) that by mutation or altered context of expression become transforming genes. The products of transforming genes cause inappropriate cell growth. More than twenty different normal cellular genes can become oncogenes by genetic alteration. Transformed cells differ from normal cells in many ways, including cell morphology, cell-to-cell interactions, membrane content, cytoskeletal structure, protein secretion, gene expression and mortality (transformed cells can grow indefinitely).
All of the various cell types of the body can be transformed into benign or malignant tumor cells. The most frequent tumor site is lung, followed by colorectal, breast, prostate, bladder, pancreas and then ovary. Other prevalent types of cancer include leukemia, central nervous system cancers, including brain cancer, melanoma, lymphoma, erythroleukemia, uterine cancer and head and neck cancer.
Cancer is now primarily treated with one, or a combination, of three types of therapies: surgery, radiation and chemotherapy. However, results with these therapies, while beneficial in some cancers, have had only marginal or no effect in many others. Furthermore, these therapies often are associated with unacceptable toxicity.
Both radiation and surgery suffer from the same theoretical drawback. It has been recognized that, given that a single malignant cell can give rise to sufficient progeny to kill the host, the entire population of neoplastic cells must be eradicated. See generally, Goodman and Gilman The Pharmacological Basis of Therapeutics (Pergamon Press, 8th Edition) (pp. 1202-1204). This concept of “total cell kill” implies that total excision of a tumor is necessary for a surgical approach, and complete destruction of all cancer cells is needed in a radiation approach, if one is to achieve a cure. In practice, this is rarely possible; indeed, where there are metastases, it is impossible.
The term “chemotherapy” simply means the treatment of disease with chemical substances. The father of chemotherapy, Paul Ehrlich, imagined the perfect chemotherapeutic as a “magic bullet;” such that the chemotherapeutic would kill invading organisms without harming the host. This target specificity is sought in all types of chemotherapeutics, including anticancer agents.
Target specificity, however, has been the major problem with anticancer agents. In the case of anticancer agents, the drug needs to distinguish between host cells that are cancerous and host cells that are not cancerous. The vast bulk of anticancer drugs are indiscriminate at this level. Typically, anticancer agents have negative hematological effects (e.g., cessation of mitosis and disintegration of formed elements in marrow and lymphoid tissues), and immunosuppressive action (e.g., depressed cell counts), as well as a severe impact on epithelial tissues (e.g., intestinal mucosa), reproductive tissues (e.g., impairment of spermatogenesis) and the nervous system. See, P. Calabresi and B. A. Chabner, In: Goodman and Gilman The Pharmacological Basis of Therapeutics (Pergamon Press, 8th Edition) (pp. 1209-1216).
Although a number of chemotherapeutic agents have been identified and are currently used for the treatment of cancer, new agents are sought that are efficacious and which exhibit low toxicity toward healthy cells.
2.2 Melanoma
Melanomas are malignant neoplasms which are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells (“Cellular and Molecular Immunology” (1991) (eds) Abbas A. K., Lichtman, A. H., Pober, J. S.; W. B. Saunders Company, Philadelphia: pages 340-341). Melanomas arise most commonly in the skin of any part of the body, or in the eye, and, rarely, in the mucous membranes of the genitalia, anus, oral cavity or other sites.
Melanocytes, which are the pigment producing cells of the epidermis, undergo malignant transformation in malignant melanoma. Through their numerous dendritic processes, melanocytes contact multiple keratinocytes, the predominant cell type of the epidermis. The adhesion molecule E-cadherin mediates the contact between the keratinocytes and the melanocytes. I. T. Valyi-Nagy, et al., Lab Invest., 69:152-9 (1993); A. Tang, et al., J. Cell. Sci., 107:983-92 (1994).
In normal skin, melanocytes are restricted to the basal layer of the epidermis, however, in malignant melanoma, melanoma cells grow throughout all layers of the epidermis, as well as in the underlying dermis. The acquisition of invasiveness is almost always accompanied by the down-regulation of E-cadherin, which is a tumor invasion suppressor. S. Vermeulen, et al., Pathol. Res. Pract., 192: 694-707 (1996). Moreover, loss of contact with keratinocytes causes melanocytes to dedifferentiate and to express melanoma-associated cell-surface antigens. I. M. Shih, et al. Am. J. Pathol., 145: 837-45 (1994).
Melanomas, which make up approximately three percent of all skin cancers, are the leading cause of death from any skin disease. Further, the worldwide increase in melanoma is unsurpassed by any other neoplasm with the exception of lung cancer in women (“Cellular and Molecular Immunology” (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J. S.; W. B. Saunders Company Philadelphia pages: 340-342; Kirkwood and Agarwala (1993) Principles and Practice of Oncology 7:1-16). Even when melanoma is apparently localized to the skin, up to 30% of the patients will develop systemic metastasis and the majority will die (Kirkwood and Agarwala (1993) Principles and Practice of Oncology 7:1-16).
Over the past four decades, the incidence of melanoma has been increasing at a higher rate than any other type of cancer. In the Connecticut Registry, between 1935 and 1939, the incidence of melanoma was 1.2/105 persons/year; this increased to 4.8/105 persons/year in 1965-1969, to 7.2/105 persons/year in 1976-1977 and to 9/105 persons/year in 1979-1980. By the year 2000, one in 90 Caucasians in the United States is expected to develop the disease (Rigel et al., 1987, J. Am. Acad. Dermatol. 17:1050-1053). In addition, due to the depletion of the Earth's ozone layer, the Environmental Protection Agency has estimated an annual increase of 2 million cases of melanoma by the year 2050. While an increasing proportion of melanomas are diagnosed sufficiently early to respond to surgical treatment and achieve a greater than 90% ten-year survival rate, it is estimated that greater than 7,000 individuals suffering from metastatic melanoma will die in the United States each year.
Melanomas are highly variable with respect to aberrant gene expression and chromosomal lesions but share a common characteristic of an acquired independence from environmental growth factors that are needed for proliferation of normal melanocytes (Halaban, 1991, Cancer Metastasis Rev. 10:129-140). In normal melanocyte proliferation as well as uncontrolled melanoma growth, receptors with tyrosine kinase activity, such as certain growth factor receptors, appear to play an important role (Id.; Becker et al., 1992, Oncogene 7:2303-2313). Various studies have suggested that a number of growth factors may be involved in melanomagenesis (Kock et al., 1991, Cancer Treat. Res. 54:41-66; Rodeck and Herlyn, 1991, Cancer Metastasis Rev. 10:89-101; Rodeck et al., 1991, J. Invest. Dermatol. 97:20-26); such growth factors include basic fibroblast growth factor (Albino et al., 1991, Cancer Res. 51:4815-4820; Rodeck and Herlyn, 1991, Cancer Metastasis Rev. 10:89-101; Dotto et al., 1989, J. Cell Biol. 109:3115-3128; contradicted by Yamanishi et al., 1992, Cancer Res. 52:5024-5029); transforming growth factors alpha and beta (Albino et al., 1991, Cancer Res. 51:4815-4820; Rodeck and Herlyn, 1991, Cancer Metastasis Rev. 10:89-101); hepatocyte growth factor/scatter factor (Halaban et al., 1992, Oncogene 7:2195-2206); tumor necrosis factor alpha and/or beta (Kimbauer et al., 1992, J. Invest. Dermatol. 98:320-326; Krutmann et al., 1992, J. Invest. Dernatol. 98:923-928); platelet derived growth factor (Rodeck and Herlyn, 1991, Cancer Metastasis Rev. 10:89-101); and various interleukins (Kimbauer et al., 1992, J. Invest. Dermatol. 98:320-326; partly contradicted by Lu et al., 1992, Proc. Natl. Acad. Sci. 89:9215-9219).
For patients with metastatic melanoma not amenable to surgical extirpation, treatment options are limited. 5-(3,3-Dimethyl-1-triazenyl)-1-H-imidaz-ole-4-carboxamide (dacarbazine, DTIC) is the most efficacious single chemotherapeutic agent for melanoma, having an overall response rate of 24%. But the duration of response to DTIC is generally quite poor. Combination therapy with other synthetic and recombinant agents, including N,N′-bis(2-chloroethyl)-N-nitrosurea (carmustine, BCNU), cisplatin, tamoxifen, interferon-alpha (INF-α) and interleukin-2 (IL-2), has a higher response rate (e.g., 30-50%) in some trials, but a durable complete response rate is uncommon and toxicity is increased. Sequential chemotherapy has promise, but, clearly, current treatment options for individuals suffering from metastatic melanoma are unsatisfactory.
Various drugs derived from natural products, such as adriamycin (doxorubicin) derivatives, bleomyciin, etoposide and vincristine, and their derivatives, have been tested for efficacy against melanoma either as single agents or in combination therapy. However, similar to the synthetic and recombinant compounds, these compounds exhibit low response rates, transient complete responses and high toxicities.
Thus, the literature is diverse and occasionally contradictory regarding the genesis and progression of melanoma, as well as for the treatment of melanomas. Furthermore, it is unclear what factors are involved in the initiation of events which lead to melanoma, as opposed to those operative in the progression of disease.
2.2.1 Endothelins
The vascular endothelium releases a variety of vasoactive substances, including the endothelium-derived vasoconstrictor peptide, endothelin (ET) (see, E., Vanhoutte et al. (1986) Annual Rev. Physiol. 48: 307-320; Furchgott and Zawadski (1980) Nature 288: 373-376). ET, which was originally identified in the culture supernatant of porcine aortic endothelial cells (see, Yanagisawa et al. (1988) Nature 332: 411-415), is a potent twenty-one amino acid peptide vasoconstrictor. It is one of the most potent vasopressors known and is produced by numerous cell types, including the cells of the endothelium, trachea, kidney and brain. ET is synthesized as a two hundred and three amino acid precursor, preproendothelin, that contains a signal sequence which is cleaved by an endogenous protease to produce a thirty-eight (human) or thirty-nine (porcine) amino acid peptide. This intermediate, referred to as big ET, is processed in vivo to the mature biologically active form by a putative ET-converting enzyme (ECE) that appears to be a metal-dependent neutral protease (see, e.g., Kashiwabara et al. (1989) FEBS Lttrs. 247: 73-76). Cleavage of big ET is required for induction of physiological responses (see, e.g., von Geldern et al. (1991) Peptide Res. 4: 32-35). In porcine aortic endothelial cells, the thirty-nine amino acid big ET intermediate is hydrolyzed at the Trp.21-Val.22 bond to generate ET-1 and a C-terminal fragment. A similar cleavage occurs in human cells from a thirty-eight amino acid intermediate. Three distinct ET isopeptides, ET-1, ET-2 and ET-3, that exhibit potent vasoconstrictor activity, have been identified.
The family of the three isopeptides, ET-1, ET-2 and ET-3 are encoded by a family of three genes (see, Inoue et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2863-2867; see, also Saida et al. (1989)J. Biol. Chem. 264: 14613-14616). The nucleotide sequences of the three human genes are highly conserved within the region encoding the mature 21 amino acid peptides and the C-terminal portions of the peptides are identical.
Release of ET from cultured endothelial cells is modulated by a variety of chemical and physical stimuli and appears to be regulated at the level of transcription and/or translation. Expression of the gene encoding ET-1 is increased by chemical stimuli, including adrenaline, thrombin and Ca2+ ionophore. The production and release of ET from the endothelium is stimulated by angiotensin II, vasopressin, endotoxin, cyclosporine and other factors (see, Brooks et al. (1991) Eur. J. Pharm. 194:115-117), and is inhibited by nitric oxide. Endothelial cells appear to secrete short-lived endothelium-derived relaxing factors (EDRF), including nitric oxide or a related substance (Palmer et al. (1987) Nature 327: 524-526), when stimulated by vasoactive agents, such as acetylcholine and bradykinin. ET-induced vasoconstriction also is attenuated by atrial natriuretic peptide (ANP).
The ET peptides exhibit numerous biological activities in vitro and in vivo. ET provokes a strong and sustained vasoconstriction in vivo in rats and in isolated vascular smooth muscle preparations; it also provokes the release of eicosanoids and endothelium-derived relaxing factor (EDRF) from perfused vascular beds. Intravenous administration of ET-1 and in vitro addition to vascular and other smooth muscle tissues produce long-lasting pressor effects and contraction, respectively (see, E., Bolger et al. (1991) Can. J. Physiol. Pharmacol. 69: 406-413). In isolated vascular strips, for example, ET-1 is a potent (EC50=4×10−10 M), slow acting, but persistent, contractile agent. In vivo, a single dose elevates blood pressure in about twenty to thirty minutes. ET-induced vasoconstriction is not affected by antagonists to known neurotransmitters or hormonal factors, but is abolished by calcium channel antagonists. The effect of calcium channel antagonists, however, is most likely the result of inhibition of calcium influx, since calcium influx appears to be required for the long lasting contractile response to ET.
ET-1, which also is secreted by keratinocytes, stimulates proliferation, chemotaxis and pigment production in melanocytes and melanoma cells. G. Imokawa, et al., Biochem. J., 314:305-12 (1996). Moreover, ultraviolet irradiation (UVR), which is implicated in melanoma development, induces a marked increase of ET-1 secretion by keratinocytes. G. Imokawa, et al., J. Biol. Chem., 267; 24675-80 (1992).
ET also mediates renin release and induces a positive inotropic action in guinea pig atria. In the lung, ET-1 acts as a potent bronchoconstrictor (Maggi et al. (1989) Eur. J. Pharmacol. 160: 179-182). ET increases renal vascular resistance, decreases renal blood flow and decreases glomerular filtrate rate. It is a potent mitogen for glomerular mesangial cells and invokes the phosphoinoside cascade in such cells (Simonson et al. (1990) J. Clin. Invest. 85: 790-797).
There are specific high affinity binding sites (dissociation constants in the range of 2-6×10−10 M) for the ETs in the vascular system and in other tissues, including the intestine, heart, lungs, kidneys, spleen, adrenal glands and brain. Binding is not inhibited by catecholamines, vasoactive peptides, neurotoxins or calcium channel antagonists. ET binds and interacts with receptor sites that are distinct from other autonomic receptors and voltage dependent calcium channels. Competitive binding studies indicate that there are multiple classes of receptors with different affinities for the ET isopeptides. The sarafotoxins, a group of peptide toxins from the venom of the snake Atractaspis eingadensis that cause severe coronary vasospasm in snake bite victims, have structural and functional homology to ET-1 and bind competitively to the same cardiac membrane receptors (Kloog et al. (1989) Trends Pharmacol. Sci. 10: 212-214).
Two distinct ET receptors, designated ETA and ETB, have been identified and DNA clones encoding each receptor have been isolated (Arai et al. (1990) Nature 348: 730-732; Sakurai et al. (1990) Nature 348: 732-735). Based on the amino acid sequences of the proteins encoded by the cloned DNA, it appears that each receptor contains seven membrane spanning domains and exhibits structural similarity to G-protein-coupled membrane proteins. Messenger RNA encoding both receptors has been detected in a variety of tissues, including heart, lung, kidney and brain. ET-1 binds with equal affinity to both ET receptors. H. Y. Kang, et al., Pfiugers Arch., 435:350-6 (1998).
The distribution of receptor subtypes is tissue specific (Martin et al. (1989) Biochem. Biophys. Res. Commun. 162: 130-137). ETA receptors appear to be selective for L. ET-1 and are predominant in cardiovascular tissues. ETB receptors are predominant in noncardiovascular tissues, including the central nervous system and kidney, and interact with the three ET isopeptides (Sakurai et al. (1990) Nature 348: 732-734). In addition, ETA receptors occur on vascular smooth muscle, are linked to vasoconstriction and have been associated with cardiovascular, renal and central nervous system diseases; whereas ETB receptors are located on the vascular endothelium, linked to vasodilation (Takayanagi et al. (1991) FEBS Lttrs. 282: 103-106) and have been associated with bronchoconstrictive disorders. Moreover, both ET receptors are expressed by melanocytes, while most melanomas express only ETB.
By virtue of the distribution of receptor types and the differential affinity of each isopeptide for each receptor type, the activity of the ET isopeptides varies in different tissues. For example, ET-1 inhibits 125I-labeled ET-1 binding in cardiovascular tissues forty to seven hundred times more potently than ET-3. 125I-labeled ET-1 binding in non-cardiovascular tissues, such as kidney, adrenal gland, and cerebellum, is inhibited to the same extent by ET-1 and ET-3, which indicates that ETA receptors predominate in cardiovascular tissues and ETB receptors predominate in non-cardiovascular tissues.
ET plasma levels are elevated in certain disease states (see, e.g., International PCT application WO 94/27979, and U.S. Pat. No. 5,382,569). ET-1 plasma levels in healthy individuals, as measured by radioimmunoassay (RIA), are about 0.26-5 pg/ml. Blood levels of ET-1 and its precursor, big ET, are elevated in shock, myocardial infarction, vasospastic angina, kidney failure and a variety of connective tissue disorders. In patients undergoing hemodialysis or kidney transplantation or suffering from cardiogenic shock, myocardial infarction or pulmonary hypertension levels as high as 35 pg/ml have been observed (see, Stewart et al. (1991) Annals Internal Med. 114: 464-469). Because ET is likely to be a local, rather than a systemic, regulating factor, it is probable that the levels of ET at the endothelium-smooth muscle interface are much higher than circulating levels.
Elevated levels of ET also have been measured in patients suffering from ischemic heart disease (Yasuda et al. (1990) Amer. Heart J. 119:801-806, Ray et al. (1992) Br. Heart J. 67:383-386). Circulating and tissue ET immunoreactivity is increased more than twofold in patients with advanced atherosclerosis (Lerman et al. (1991) New Engl. J. Med. 325:997-1001). Increased ET immunoreactivity also has been associated with Buerger's disease (Kanno et al. (1990) J. Amer. Med. Assoc. 264:2868) and Raynaud's phenomenon (Zamora et al. (1990) Lancet 336 1144-1147). Increased circulating ET levels were observed in patients who underwent percutaneous transluminal coronary angioplasty (PTCA) (Tahara et al. (1991) Metab. Clin. Exp. 40:1235-1237; Sanjay et al. (1991) Circulation 84 (Suppl. 4):726), and in individuals (Miyauchi et al. (1992) Jpn. J. Pharmacol. 58:279 P; Stewart et al. (1991) Ann. Internal Medicine 114:464-469) with pulmonary hypertension.
2.2.1.1 Endothelin Agonists and Antagonists
Because ET is associated with certain disease states and is implicated in numerous physiological effects, compounds that can interfere with or potentiate ET-associated activities, such as ET-receptor interaction and vasoconstrictor activity, are of interest. Compounds that exhibit ET antagonistic activity have been identified. For example, a fermentation product of Streptomyces misakiensis, designated BE-18257B, has been identified as an ETA receptor antagonist. BE-18257B is a cyclic pentapeptide, cyclo(D-Glu-L-Ala-allo-D-lle-L-Leu-D-Trp), which inhibits 125I-labeled ET-1 binding in cardiovascular tissues in a concentration-dependent manner (IC50 1.4 μM in aortic smooth muscle, 0.8 μM in ventricle membranes and 0.5 μM in cultured aortic smooth muscle cells), but fails to inhibit binding to receptors in tissues in which ETB receptors predominate at concentrations up to 100 μM. Cyclic pentapeptides related to BE-18257B, such as cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), have been synthesized and shown to exhibit activity as ETA receptor antagonists (see, U.S. Pat. No. 5,114,918 to Ishikawa et al.; see, also, EP A10 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991)). Studies that measure the inhibition by these cyclic peptides of ET-1 binding to ET-specific receptors indicate that these cyclic peptides bind preferentially to ETA receptors. Other peptide and non-peptidic ETA antagonists have been identified (see, e.g., U.S. Pat. Nos. 5,352,800, 5,334,598, 5,352,659, 5,248,807, 5,240,910, 5,198,548, 5,187,195, 5,082,838). These include other cyclic pentapeptides, acyltripeptides, hexapeptide analogs, certain antraquinone derivatives, indanecarboxylic acids, certain N-pyriminylbenzenesulfonamides, certain benzenesulfonamides and certain naphthalenesulfonamides (Nakajima et al. (1991) J. Antibiot. 44:1348-1356; Miyata et al. (1992) J. Antibiot. 45:74-8; Ishikawa et al. (1992) J. Med. Chem. 35:2139-2142; U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A10 569 193; EP A1 0 558 258; EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991); Canadian Patent Application 2,067,288; Canadian Patent Application 2,071,193; U.S. Pat. No. 5,208,243; U.S. Pat. No. 5,270,313; U.S. Pat. No. 5,464,853; Cody et al. (1993) Med. Chem. Res. 3:154-162; Miyata et al. (1992) J. Antibiot 45:1041-1046; Miyata et al. (1992) J. Antibiot 45:1029-1040, Fujimoto et al. (1992) FEBS Lett. 305:41-44; Oshashi et al. (1002) J. Antibiot 45:1684-1685; EP A10 496 452; Clozel et al. (1993) Nature 365:759-761; International Patent application WO93/08799; Nishikibe et al (1993) Life Sci. 52:717-724; and Benigni et al. (1993) Kidney Int. 44:440-444). In general, the identified compounds have activities in in vitro assays as ETA antagonists at concentrations on the order of about 50-100 μM or less. A number of such compounds have also been shown to possess activity in in vivo animal models. Very few selective ETB antagonists have been identified.
2.2.1.2 Endothelin Antagonists and Agonists as Therapeutic Agents
It has been recognized that compounds that exhibit activity at IC50 or EC50 concentrations on the order of 10−4 or lower in standard in vitro assays that assess ET antagonist or agonist activity have pharmacological utility (see, e., U.S. Pat. Nos. 5,352,800, 5,334,598, 5,352,659, 5,248,807, 5,240,910, 5,198,548, 5,187,195, 5,082,838). By virtue of this activity, such compounds are considered to be useful for the treatment of hypertension such as peripheral circulatory failure, heart disease such as angina pectoris, cardiomyopathy, arteriosclerosis, myocardial infarction, pulmonary hypertension, vasospasm, vascular restenosis, Raynaud's disease, cerebral stroke such as cerebral arterial spasm, cerebral ischemia, late phase cerebral spasm after subarachnoid hemorrhage, asthma, bronchoconstriction, renal failure, particularly post-ischemic renal failure, cyclosporine nephrotoxicity such as acute renal failure, colitis, as well as other inflammatory diseases, endotoxic shock caused by or associated with ET, and other diseases in which ET has been implicated.
In view of the numerous physiological effects of ET and its association with certain diseases, ET is believed to play a critical role in these pathophysiological conditions (see, e., Saito et al. (1990) Hypertension 15: 734-738; Tomita et al. (1989) N. Engl. J. Med. 321: 1127; Kurihara et al. (1989) J. Cardiovasc. Pharmacol. 13(Suppl. 5): S 13-S 17; Doherty (1992) J. Med. Chem. 35: 1493-1508; Morel et al. (1989) Eur. J. Pharmacol. 167: 427-428). More detailed knowledge of the function and structure of the ET peptide family should provide insight in the progression and treatment of such conditions.
To aid in gaining further understanding of and to develop treatments for ET-mediated or related disorders, there is a need to identify compounds that modulate or alter ET activity. Identification of compounds that modulate ET activity, such as those that act as specific antagonists or agonists, may not only aid in elucidating the function of ET, but may yield therapeutically useful compounds. In particular, compounds that specifically interfere with the interaction of ET peptides with the ETA or ETB receptors should be useful in identifying essential characteristics of ET peptides, should aid in the design of therapeutic agents and may be useful as disease specific therapeutic agents.
2.2.2 Cadherins
In vivo, cell-cell adhesion plays an important role in a wide range of events including morphogenesis and organ formation, modulation of the immune system, the formation of cell junctions and tumor metastasis and invasion. Additionally, cell-cell adhesion is crucial for the maintenance of tissue integrity, e.g., of the intestinal epithelial barrier, of the blood brain barrier and of cardiac muscle.
Intercellular adhesion is mediated by specific cell adhesion molecules. Cell adhesion molecules have been classified into at least three superfamilies including the immunoglobulin (Ig) superfamily, the integrin superfamily and the cadherin superfamily. All cell types that form solid tissues express some members of the cadherin superfamily suggesting that cadherins are involved in selective adhesion of most cell types.
Cadherins have been described generally as glycosylated integral membrane proteins that have an N-terminal extracellular domain that determines binding specificity (the N-terminal 113 amino acids appear to be directly involved in binding), a hydrophobic membrane-spanning domain and a C-terminal cytoplasmic domain (highly conserved among the members of the superfamily) that interacts with the cytoskeleton through catenins and other cytoskeleton-associated proteins. Some cadherins lack a cytoplasmic domain, however, and appear to function in cell-cell adhesion by a different mechanism than cadherins that do have a cytoplasmic domain. The cytoplasmic domain is required for the binding function of the extracellular domain in cadherins that do have a cytoplasmic domain. Binding between members of the cadherin family expressed on different cells is mainly homophilic (i.e., a member of the cadherin family binds to cadherins of its own or a closely related subclass) and Ca2+-dependent.
The first cadherins to be described (E-cadherin in mouse epithelial cells, L-CAM in avian liver, uvomorulin in the mouse blastocyst, and CAM 120/80 in human epithelial cells) were identified by their involvement in Ca2+-dependent cell adhesion and by their unique immunological characteristics and tissue localization. With the later immunological identification of N-cadherin, which was found to have a different tissue distribution from E-cadherin, it became apparent that a new family of Ca2+-dependent cell-cell adhesion molecules had been discovered.
The molecular cloning of the genes encoding mouse E—(see Nagafuchi et al., Nature, 329: 341-343 (1987)), chicken—(Hatta et al., J. Cell Biol., 106: 873-881 (1988)), and mouse P—(Nose et al., EMBO J. 6: 3655-3661 (1987)) cadherins provided structural evidence that the cadherins comprised a family of cell adhesion molecules. Cloning of chicken L-CAM (Gallin et al., Proc. Natl. Acad. Sci. USA, 84: 2808-2812 (1987)) and mouse uvomorulin (Ringwald et al., EMBO J., 6: 3647-3653 (1987)) revealed that they were identical to E-cadherin. Comparisons of the amino acid sequences of E-, N-, and P-cadherins showed a level of amino acid similarity of about 45%-58% among the three subclasses.
The determination of the tissue expression of the various cadherins reveals that each subclass of cadherins has a unique tissue distribution pattern. For example, E-cadherin is found in epithelial tissues while N-cadherin is found in nonepithelial tissues such as neural and muscle tissue. The unique expression pattern of the different cadherins is particularly significant when the role each subclass of cadherins may play in vivo in normal events (e.g., the maintenance of the intestinal epithelial barrier) and in abnormal events (e.g., tumor metastasis or inflammation) is considered.
Suppression of cadherin function also has been implicated in the progression of various cancers. See Shimoyama et al., Cancer Res., 52: 5770-5774 (1992). In fact, E-cadherin has been shown to be a tumor invasion suppressor. M. Y. Hsu, et al., J. Investig. Dermatol. Symp. Proc., 1:188-94 (1996). Furthermore, loss of E-cadherin (membrane associated) expression was found to be correlated with: lymph node metastasis of squamous cell carcinoma (J. H. Schipper, et al., Cancer Research 1991, 51: 6328-6337); dedifferentiation of meningiomas (Y. Tohma, et al., Cancer Research, 1992, 52: 1981-1987); high Gleason grade of prostate carcinomas (R. Umbas, et al., Cancer Research, 1992, 52: 5104-5109); infiltrative growth of basal cell carcinoma (A. Pizarro, et al. Br. J Cancer, 1994, 69: 157-162); dedifferentiation and metastasis of breast carcinoma (C. Gamallo, et al., American Journal of Pathology, 1993, 142: 987-993; R. Moll, et al., American Journal of Pathology, 1993, 143: 1731-1742; H. Oka, et al., Cancer Research, 1993, 53: 1696-1701); dedifferentiation, high Dukes stage and metastasis of colon carcinoma (S. Dorudi, et al., American Journal of Pathology, 1993, 142: 981-986; A. R. Kinsela, et al., Cancer Research, 1994, 67: 904-909); poor prognosis of bladder cancer (in combination with gp78) (T. Otto, et al., Cancer Research, 1994, 54: 3120-3123); dedifferentiation of thyroid carcinoma (C. Brabant, et al., cancer Research, 1993, 53: 4987-4993); and lymph node metastasis, high grade and advanced stage of pancreatic carcinoma (M. Pignatelli, et al., Journal of Pathology, 1994, 174: 243-248).
Recently, it has been shown that E-cadherin is expressed on cultured melanocytes where it mediates adhesion to keratinocytes. Danen et al., 1996, Mel. Res., 6:127-131. Loss of contact with keratinocytes causes melanocytes to dedifferentiate and to express melanoma-associated cell-surface antigens. I. M. Shih, et al, Am. J. Pathol., 145:837-45 (1994). Further, the acquisition of invasiveness of melanocytes is almost always accompanied by the down-regulation of E-cadherin. Moreover, the expression of E-cadherin is reduced in most melanoma cell lines. Thus, E-cadherin mediated cell contact between melanocytes and keratinocytes may be critical for the maintenance of normal melanocyte phenotype.
2.2.2.1 Catenins
The catenins have been classified into α, β and γ on the basis of their electrophoretic mobilities (The EMBO Journal, 8, p 1711-1717 (1989)). Catenins are cytoplasmic proteins that are critical for E-cadherin function in cellular adhesion. J. M. Daniel, et al., Bioessays, 19:883-91 (1997). They bind to the cytoplasmic region of cadherins and function to modulate adhesion and/or bridge cadherins to the actin cytoskeleton. The catenins transmit an adhesion signal and anchor the cadherin to the actin cytoskeleton. The classical cadherins, E, N and P, bind directly to β-catenin. These, in turn, associate with the vinculin-like protein α-catenin, which is thought to link cadherin complexes to the actin cytoskeleton, either by direct interaction or indirectly via α-actinin. Daniel et al., 1997, BioEssays, 19(10): 883-891. Thus, disruption of the cadherin/catenin function could be integral to numerous diseases associated with a decrease in cadherin binding.
2.2.2.2 Caspases
Caspases, which are proteases best known for their role in apoptotic cell death, also are known to participate in inflammatory processes. Several studies recently have shown that proteases belonging to the caspase family are capable of cleaving β-catenin with a concomitant down-regulation of E-cadherin.
Caspases are activated in a sequential cascade beginning with apical caspases, such as caspase-8, which then activate distal caspases, such as caspases-3 and 7, which execute apoptotic cell death through cleavage of a variety of critical cell substrates. Caspase-8 may directly cleave catenin proteins or activate other as of yet unidentified caspase(s) which cleave catenin proteins, and this cleavage likely leads to destabilization and disruption of E-cadherin:catenin complexes at the plasma membrane. Indeed, caspase cleavage has been shown to prevent interaction of β-catenin with α-catenin, the latter of which serves to anchor the E-cadherin:catenin complex to the actin cytoskeleton.
Thus, caspase activation may play a role in the down-regulation and destabilization of cadherin:catenin complexes. Moreover, as discussed above, down-regulation of cadherin-catenin complexes may play a role in the progression of various cancers.